Role of Toll-like Receptors in Spontaneous Commensal-Dependent Colitis Seth Rakoff-Nahoum, Liming Hao, Ruslan Medzhitov  Immunity  Volume 25, Issue 2, Pages 319-329 (August 2006) DOI: 10.1016/j.immuni.2006.06.010 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Absence of MyD88 Prevents Gross Pathological Changes in IL-10- but Not IL-2-Deficient Mice Photographs of representative mesenteric lymph nodes, spleens, and colons from wt, Myd88−/−, Il10−/−, Il10−/−Myd88−/−, Il2−/−, and Il2−/−Myd88−/− mice. Immunity 2006 25, 319-329DOI: (10.1016/j.immuni.2006.06.010) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 The Development of Spontaneous Colitis in the Absence of IL-10, but Not IL-2, Is Completely Dependent on Signaling through MyD88 (A) Histopathological scoring of colons of wt (n = 19; ranging from 16 weeks to 1.5 years old), Myd88−/− (n = 11; ranging from 16 weeks to 1.5 years of age), Il10−/− (n = 13; ranging from 16 to 22 weeks of age), Il10−/−Myd88−/− (n = 19; ranging from 16 weeks to 1.5 years of age), Il2−/− (n = 19; ranging from 16 to 24 weeks of age), and Il2−/−Myd88−/− (n = 12; ranging from 16 to 24 weeks of age) mice. Scores are based on epithelial hyperplasia and mononuclear and polymorphonuclear infiltrate of the proximal, middle, and distal colon. (B) Representative photomicrographs (magnifications ×40 and ×100; haemotoxylin and eosin staining) of colons from wt, Myd88−/−, Il10−/−, Il10−/−Myd88−/−, Il2−/−, and Il2−/−Myd88−/− mice. Immunity 2006 25, 319-329DOI: (10.1016/j.immuni.2006.06.010) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Regulation of MyD88-Dependent and -Independent T Cell Activation and DC Migration by IL-10 and IL-2 Mesenteric lymph node cells from wt, Myd88−/−, Il10−/−, Il10−/−Myd88−/−, Il2−/−, and Il2−/−Myd88−/− mice were stained for (A) CD4 and CD25 and (B) CD4 and CD44. (C) % CDllc+ MHC ClassIIhi, (D) total number of CDllc+ MHC ClassIIhi, and (E) total number of cells per animal isolated from mesenteric (left) and axillary (right) lymph nodes. Data are the percentage of live cells as determined by forward and side scatter. Data are of four individual experiments with 2–4 mice per genotype per experiment. Error bars are ±SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (compared to wt) according to the Student's test. Immunity 2006 25, 319-329DOI: (10.1016/j.immuni.2006.06.010) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 MyD88 Is Required for the Generation of Pathologic Th1 Responses in the Absence of IL-10, but Not IL-2 Lamina propria mononuclear cells were isolated from the colons of wt, Myd88−/−, Il10−/−, Il10−/−Myd88−/−, Il2−/−, and Il2−/−Myd88−/− mice. Freshly isolated cells were (A) cultured with plate bound anti-CD3 antibody at 2 × 106 cells/ml for 48 hr, after which supernatants were collected and assayed for the presence of IFN-γ by ELISA or (B and C) assayed for intracellular cytokine production of IFNγ and TNF. Total number of cytokine-positive LPMC intestine {% cytokine+ × average number of LPMC per genotype in pooled experiment}. Data are of two individual experiments in which 2–4 colons per genotype were pooled. Error bars are ±SEM. ∗p < 0.05, ∗∗p < 0.01 (compared to wt) according to the Student's test. Immunity 2006 25, 319-329DOI: (10.1016/j.immuni.2006.06.010) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 IL-12- or -23-Independent Intestinal Th1 Pathology in the Absence of IL-2 (A) Whole colons of wt, Myd88−/−, Il10−/−, Il10−/−Myd88−/−, Il2−/−, and Il2−/−Myd88−/− mice were cultured for 24 hr in serum-free media. Concentration of IL-12 or -23 p40 in supernantant was determined by ELISA. n = 5–8 mice per genotype. (B) RNA was prepared from isolated colonic lamina propria mononuclear cells of wt, Myd88−/−, Il10−/−, Il10−/−Myd88−/−, Il2−/−, and Il2−/−Myd88−/− mice. cDNA was prepared and quantitative polymerase chain reaction was performed. Expression of IL-27 p28 and IL-27 EBI3 was normalized to HPRT and relative induction compared to colonic LPMC from wt mice. Data are of 5 individual experiments of 4–5 mice per genotype. Error bars are ±SEM. ∗∗p < 0.01, ∗∗∗p < 0.001 (compared to wt) according to the Student's test. Immunity 2006 25, 319-329DOI: (10.1016/j.immuni.2006.06.010) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Commensal-Dependent Colitis in the Absence of IL-2 Is Completely Independent of TLR Signaling (A) Representative microphotos of large intestine from Myd88−/− Trif−/−, Il2−/−, Il2−/−Myd88−/−, Il2−/−Myd88−/− Trif−/− age-matched littermates. Magnification ×200. (B and C) IFNγ (B) and IL-27 p28 (C) and EBI3 expression in colons. RNA was prepared from colons of wt, Myd88−/−, Trif−/−, Trif−/−Myd88−/−, Il2−/−, Il2−/−Myd88−/−, and Il2−/−Myd88−/−TRIF−/− mice. cDNA was prepared and quantitative polymerase chain reaction was performed. Expression of IFNγ, IL-27 p28, and EBI3 was normalized to HPRT and relative induction compared to colons from wt mice. Data are of duplicate experiments of 2–4 mice per genotype. Error bars are ±SEM. ∗∗p < 0.01 (compared to wt) according to the Student's test. Immunity 2006 25, 319-329DOI: (10.1016/j.immuni.2006.06.010) Copyright © 2006 Elsevier Inc. Terms and Conditions